Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is concerned with development of vaccines and other infection inhibitors against HPV and elucidation of the PV life cycle. We have developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, referred to hereafter as pseudovirions. This methodology represents a technical breakthrough in papillomavirus research, and we have exploited this development in several ways, as outlined below. Pseudovirus production technology is being used to explore the basic features of papillomavirus infection. We have determined that a cleavage of the terminus of L2 by the cellular proprotein convertase furin is required for papillomavirus infection. This cleavage presumably exposes a binding site for a conserved cellular receptor, because in other studies we have determined that broadly cross-type neutralizing L2 antibodies bind the peptide immediately downstream of the furin cleavage site. This cleavage site becomes exposed after engagement of the cell surface and/or extracellular matrix. The requirement for furin is the first example of a requirement of a sequence-specific cleavage of a virion protein for entry of any virus. However, this function may be an example of convergent evolution, as many bacterial toxins require furin for their escape from the endosome, and furin inhibition prevents the uncoated papillomavirus particle from escaping the endosome. These results are consistent with a collaborative study with Martin Sapps group in which we show that endosomal escape of papillomavirus capsids is L2-dependent and that a C-terminal L2 peptide has endosomal lysis activity. In studies of innate immunity to papillomaviruses, we have determined that the cationic peptide alpha defensin 5 is a potent inhibitor of papillomavirus infection. In the presence of alpha defensin 5, the capsids become trapped in a late endosome compartment. Interestingly alpha defensin 5 is normally found in cervicovaginal mucus of women in a range that can overlap the inhibitory concentration for HPV infection in vitro. The results raise the possibility that polymorphisms in the alpha defensin 5 gene may play a role in susceptibility to HPV infection. In a rather extensive screen of in vitro HPV infection inhibitors, we have determined that carrageenan, a sulfated polysccharide extrated from red algae, is an exceptionally potent broad spectrum genital HPV infection inhibitor. It is nontoxic to cells and at least 100-fold more potent than any HPV infection inhibitor previously identified. It is inexpensive and commonly added to processed foods as a stabilizer or gelling agent. It is also used as the primary gelling agent in certain sexual lubricants. Remarkably, these lubricants can inhibit genital HPV infection in vitro at more than a one million-fold dilution. We are currently working with the NCI extramural division DCP to initial clinical trial of carrageenan as a microbicide to prevent genital HPV infection in young women. We have recently developed the first cervicovaginal challenge model for HPVs. We have found the infection of the female mouse genital tract, even of monolayer endocervical cells, requires exposure the basement membrane to the virus. The capsids bind avidly to the basement membrane but not to the apical surfaces of intact columnar or stratified squamous epithelia. Using a quantitative assay based on whole tissue fluorecence imaging after infection with red fluorescent protein epressing pseudovirions, we have further determined that nonoxynol-9 (N-9)and Conceptrol, an over the counter spermacide that containing N-9, dramatically potentiate in vivo papillomavirus infection, presumably due to it's ability to permeablize the epithelial layers and thereby expose the basement membrane to the virus. Interesting, no infection was detected if nonoxynol-9 was formulated in carrageenan rather than its normal gelling agent carboxymethly cellulose. The results suggest that women using this common spermacide may be at increased risk of aquiring genital HPV infection and that this risk might be eliminated by reformulation of N-9 in a carrageenan based gel